The present invention relates to a process for producing cis-3-hydroxy-L-proline. Cis-3-hydroxy-L-proline is useful as a starting compound for medicines and an additive to foods. The present invention also relates to a novel enzyme capable of catalyzing the hydroxylation of L-proline at the 3-position of L-proline (hereinafter referred to as xe2x80x9cL-proline-3-hydroxylasexe2x80x9d). The novel enzyme is used in the above-mentioned process.
The present invention also relates to a gene encoding a protein having an activity of L-proline-3-hydroxylase (hereinafter referred to as xe2x80x9cL-proline-3-hydroxylase genexe2x80x9d) which is useful for the above-mentioned process, a transformant containing the gene, and a process for producing L-proline-3-hydroxylase using the transformant.
Heretofore, chemosynthetic methods of producing cis-3-hydroxy-L-proline are known [J. Amer. Chem. Soc., 84, 3980 (1962); J. Amer. Chem. Soc., 85, 2824 (1963); Nature 289, 310 (1981); J. Org. Chem., 54, 1866 (1989); Acta Chemica Scandinavica, 43, 290 (1989)].
The conventional chemosynthetic methods for producing cis-3-hydroxy-L-proline are not satisfactory for industrial production, because of (1) the expensive raw materials, (2) too many reaction steps, (3) the complicated procedures for isolating and purifying the product and/or (4) the lower productivity of cis-3-hydroxy-L-proline.
No chemosynthetic or biological method of producing cis-3-hydroxy-L-proline by hydroxylating L-proline both regio-selectively and stereo-selectively, has been reported.
The object of the present invention is to provide an advantageous process for the production of cis-3-hydroxy-L-proline which is industrially applicable, and a second object of the present invention is to provide a novel enzyme which catalyzes the hydroxylation of L-proline at the 3-position of L-proline and which is useful in the above process.
The present invention provides a process for producing cis-3-hydroxy-L-proline, which comprises allowing L-proline to coexist with 2-ketoglutaric acid, a divalent iron ion and an enzyme source which catalyzes the hydroxylation of L-proline at the 3-position of L-proline in an aqueous medium to convert L-proline into cis-3-hydroxy-L-proline, and recovering the cis-3-hydroxy-L-proline from the aqueous medium.
The present invention further provides a novel hydroxylase (L-proline-3-hydroxylase) having the following physicochemical properties:
(1) Action and Substrate Specificity:
The enzyme catalyzes the hydroxylation of L-proline at the 3-position of L-proline in the presence of 2-ketoglutaric acid and a divalent iron ion to produce cis-3-hydroxy-L-proline.
(2) Optimum pH Range:
The enzyme has an optimum pH range of 6.5 to 7.5, for its reaction at 30xc2x0 C. for 20 minutes.
(3) Stable pH Range:
The enzyme is stable at pH values of 6.5 to 8.0, when it is allowed to stand at 4xc2x0 C. for 23 hours.
(4) Optimum Temperature Range:
The optimum temperature range is 35 to 40xc2x0 C. when it is allowed to stand at pH 7.0 for 15 minutes.
(5) Stable Temperature Range:
The enzyme is inactivated, when it is allowed to stand at pH 7.5 and at 50xc2x0 C. for 30 minutes.
(6) Inhibitors:
The activity of the enzyme is inhibited by metal ions of Zn++, Cu++, Co++ and Ba++ and ethylenediaminetetraacetic acid (EDTA).
(7) Activation:
The enzyme does not need any cofactor for its activation. L-Ascorbic acid accelerates the activity of the enzyme.
(8) Km Value:
The Km value is 0.49 mM for L-proline and is 0.11 mM for 2-ketoglutaric acid, when determined in a 100 mM N-tris(hydroxymethyl)methyl-2-aminoethansulfonic acid (TES) buffer (pH 7.0) containing 5 mM L-ascorbic acid, 1 mM ferrous sulfate and a pre-determined amount of this enzyme.
(9) Isoelectric point;
The enzyme has an isoelectric point of 4.3 as determined by the Phast system.
(10) Molecular Weight:
The enzyme has a molecular weight of 35,000xc2x15,000 daltons as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis.
(11) N-terminal Amino Acid Sequence:
The enzyme has an N-terminal amino acid sequence illustrated by SEQ ID NO: 5.
The present invention provides an L-proline-3-hydroxylase gene and a transformant containing the gene for the purpose of producing cis-3-hydroxy-L-proline efficiently and industrially using L-proline-3-hydroxylase from L-proline that is available at low cost, a process for mass-producing the L-proline-3-hydroxylase using the transformant containing the gene, and a process for producing cis-3-hydroxy-L-proline industrially at low cost using a transformant containing the gene conding for L-proline-3-hydroxylase.